Unit One Instruments

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Analytical techniques

UNIT 1


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Self Study

Principle of the followinginstruments/techniques and identify specificanalytes that are measured by each

instrument: Flourometry

Turbidimetry

Nephelometry Chemiluminescence

Chromotography (HPLC; GLC & TLC)

Elisa Prof T. Matsha 2


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Lecture Outline

Photometry & Spectrophotometry Mass spectrophotometry

Electrochemistry (Nersnt equation) Electrophoresis

Osmometry

Enzyme kinetics

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Photometry &Spectrophotometry (1)

Measurement of light intensity

Light as other forms of electromagneticradiation make characteristic patterns

(waves) as they travel through space Wavelength distance between two peaks

(high points) as the light travels in a wavelike manner
http://id.wikipedia.org/wiki/Berkas:Wavelength.pnghttp://id.wikipedia.org/wiki/Berkas:Wavelength.png


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Photometry &

Spectrophotometry (2)

Each wave has a certain shape and

length depending on the frequency ofthe waves. Frequency of a wave is inversely proportional

to the wavelength


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Previously, photometric instruments measured lightintensity independently of wavelength

Sunlight mixture of spectrum of radiant energy atdifferent wavelengths (rainbow) human eye recognizes itas white

Modern Instruments can isolate a narrow wavelengthrange of the spectrum for measurements

Filters filter photometers Prisms or gratings - spectrophotometers
http://www.lpi.usra.edu/education/fieldtrips/2005/activities/ir_spectrum/images/emspectrum.jpg


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Determinations in the ClinicalLaboratory are based on measurementof radiant energy

Emitted Partially reflected Transmitted

Absorbed Photometers light emitted Spectrophotometers - absorbed



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SPECTROPHOTOMETER

Used to measure to concentrationsof substances


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SPECTROPHOTOMETER cont.

A combination of a spectrometer & a photometerSpectrometer produces light of any selectedPhotometer measures light intensity

A cuvette with liquid is placed between the 2.


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The amount of light passing through the cuvetteis measured by the photometer.

The photometer delivers a voltage signalto a display device.The signal changes as the amount of lightabsorbed by the liquid changes


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Lightabsorbed

Transmitted

light


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Light absorbed

Units: Absorbance (A) or Optical density (OD)

(logarithmic scale)

Transmitted light - % transmission(Arithmetic scale)


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Obeys Beers law:

If a solute absorbs light of a particular ,

the absorbance is directly proportional tothe concentration of substance in solution.

I0A = log = clI


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Beers Law (1)

Beers law The intensity of a solution when viewed through monochromatic light

(single wavelength, e.g. 600nm) is directly proportional to theconcentration of the substance through which the light passes.

Lamberts law

- When monochromatic light passes through a transparent medium, therate decrease in intensity with the thickness of the medium isproportional to the intensity of light

Beer-Lambert law (also known as Beers law)- When monochromatic light passes through a coloured solution the

amount of light transmitted decreases exponentially with the increasein concentration of the solution through which the light passes orsimply; absorbance is directly related to concentration if the light pathstay constant


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Beers Law (2)

Absorbance Absorption is a process in which incident radiated

energy is retained without reflection or transmissionon passing through a medium

Therefore for ray to be absorbed it must have thesame frequency as a rotational or vibrationalfrequency in the atom or molecule it strikes

Transmittance The ratio of transmitted energy to the amount ofincident energy is called transmittance.


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USE OF ASPECTROPHOTOMETER

Blank


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Beers Law (3)

Some of Io is either reflected by surface of the cell orabsorbed by solvent or cell wall , Io < Is

Percent transmittance %T = Io / Is X 100 For some applications in optics it might be useful to see

transmittance values as percent transmittance values. Allintensities will be scaled to fit an interval between 0 and 100percent transmittance.

Focus interest eliminate factors use blank Blank absence of compound of interest but same solvent. No

light absorbed %T = 100%

Add compound of interest serially %T varies inversely andlogarithmically with concentration


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Clinical Example

1. Blank Reading reagent buffer without serum

Read the blank Most light transmitted & small amount absored bycuvette, solvent or reflected from detector

Set instrument abitrarily at 100%T (A = 0)2. Sample Reading Reagent buffer + serum Difference amount light passed blank vs. sample due

to presence of compound measured % T Sample beam signal X 100

Blank beam signal


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1. Switch on2.Set wavelength3.Use after 30 min


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4. Calibratea. Insert blank


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5. Set to 0


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4. Insert sample &read


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Beers Law (4)

Absorbance is more convenient to usebecause it is directly proportional toconcentration.

Amount of light absorbed particularwavelength depends:1. Molecules and ions present2. [ ]

3. pH4. Temperature


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Standard Curve

Unknown [ ] isdetermined from acalibration curve orstandard curve

Standards of knownconcentration

Plot on graph linearcurve

B2MG Concentration (g/ml)

Absorbance(450nm)

0 0.046

0.625 0.385

1.25 0.723

2.5 1.241

5 2.199

10 3.094


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SpectrophotometicInstruments

Measure light transmitted by solution

Mathematically converted absorbance

Determine [ ] light absorbing substance


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Spectrophotometer (2)

Light Source Provides radiant energy

Visible light (350 - 700nm) tungsten light bulb

To increase lifetime iodine or bromide is added -tungsten-iodide lamp UV region (165 -360nm) low pressure mercury-vapor

lamp, emits discontinuous spectrum Hydrogen & deuterium lamps low. Deuterium-

discharge more stable than hydrogen Mercury & xenon high pressure


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Monochromator - isolate radiant energy of desired wavelength butexcludes others

Monochromator


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Filters

To obtain monochromatic light useTypes1. coloured-glass filter

Transmistts energy over a wide range of wavelength Not precise Simple & inexpensive

2. Interference filters Pass very narrow range wavelength Efficient
http://images.google.co.za/imgres?imgurl=http://www.tufts.edu/as/tampl/projects/micro_rs/monochromator4.jpg&imgrefurl=http://www.tufts.edu/as/tampl/projects/micro_rs/setup.html&h=307&w=345&sz=19&hl=en&start=1&tbnid=8dA7BUCxxYBM5M:&tbnh=107&tbnw=120&prev=/images%3Fq%3Dmonochromator%26gbv%3D2%26svnum%3D10%26hl%3Den%26sa%3DGhttp://images.google.co.za/imgres?imgurl=http://www.tufts.edu/as/tampl/projects/micro_rs/monochromator4.jpg&imgrefurl=http://www.tufts.edu/as/tampl/projects/micro_rs/setup.html&h=307&w=345&sz=19&hl=en&start=1&tbnid=8dA7BUCxxYBM5M:&tbnh=107&tbnw=120&prev=/images%3Fq%3Dmonochromator%26gbv%3D2%26svnum%3D10%26hl%3Den%26sa%3DG


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Prisms

Type of monochromator Separates white light to continuous spectrum

by refraction, i.e. shorter wavelengths arerefracted or bent.

Consequently nonlinear spectrum with longerwavelengths closer together, but

Suitable narrow-bandwith portion of the

spectrum


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Separation of light to differentwavelengths

Most commonly used monochromators

Consists of parallel grooves etchedpolished surface

Diffraction gratings


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Cuvettes

Cuvette/Sample/Absorptioncell:

Holds sample& provides

con stant l ight path

Round / square

Light path must be kept

constant

Otherwise A no t C


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Round

Difficult constant light path Not uniform Etched for constant positionSquare/rectangular

Plane-parallel optical surface, constant light path Less error Most common Plastic cells inexpensive, but designed for single use

application

Good clarity for both UV and visible light Problems etching by solvents, temp. deformations,

cleaning Quartz cuvettesexpensive, UV range

Cuvette types


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QC: Cuvettes

Scratched opt ical surface scatter light

Do not touchon optical surface

Wipeoptical surface tissue before use

Insert correct orientation spectrophotometer

Clean immediatelyafter use (DO NOT soak)

(mild detergent & rinse deionised water)

Drainupside down to dry


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Convert transmitted radiant energy equivalent amount electrical energy

Photodetector


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Photodetector types

Pho tocell / barr ier-layer cell

=> least expensive

=> film light-sensitive material (eg. selenium /

iron / silver)=> require no external voltage

=> rely internal e- transfer produce current

=> wide bandpass instruments


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Phototube

=> also photosensitive material=> e- generated from light energy=> outside voltage required

h l i li b


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photomultiplier tube

=> detects & amplifies radiant energy=> e- attracted series anodes / dynodes=> each (+) voltage=> generate 2ndary e-=> multiple cascade => amplification=> thus 200x more sensitive than phototube=> narrow bandpass instruments

wavelength scanner instruments

double-beam spectrophotometers


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DOUBLE BEAM


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DOUBLE-BEAMSPECTROPHOTOMETER

Automatic correctionsample A &reference A


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QA/QC Spec.

Wavelength accuracy Stray light scratched and dust particles Linearity


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Spec in Clin lab

General chemistryanalytes, e.g. glucose


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Mass spectrophotometer

MS used to:

Identify unknown compounds

[ ] of known substances

Molecular structure

Chemical composition of both in-& organic materia In clinical chemistry:

Drug metabolism

Drug abuse (steroids use in sport)

Damage to DNA

Metabolic disorders in infants

Research, e.g. search for unique proteins in specimen for use as

diagnostic or therapeutic targets

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Mass spec basic components

Sample converted into

ions or molecules by

thermal or electrical

energy

The ions in a gaseousmedium are accelerated

into the mass analyzer

where they are separated

into species, such thatdifferent species of ions

strike the detector at

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ATOMIC ABSORPTION


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ATOMIC ABSORPTIONSPECTROPHOTOMETER

Measure [ ]. by detecting A ofelectromagneticradiationby atoms

(not molecules)


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Components


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Light Source

Hallow-cathode lamp Consists of evacuated gas-tight chamber +

anode

Cathode Inert gas(argon / helium)=> voltage applied=> filler gas ionised

=> ions excite metal atoms=> light energyemitted


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Sample Cell

Flame

Why? Sample must contain reducedmetal in atomic vaporised state

Done via heat of flame break chemicalbonds unexcited atoms


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AAS vs SPEC

Light source passes through sample

Note, atoms though bonds are broken ground state (unexcited)

Light source excites atoms returns toground state emits energy = absorbedlight


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Chopper

Aim: measure light absorbed by atoms

Need to distinguish between lightemitted by light source and excitedatoms

Hence, the chopper


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Monochromator

Also used to protect the photodetectorfrom excessive light from flameemissions


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Adv:Disadvantages

ADV:=> very sensitive & precise

DISADV: onlymeasure elementsthat exist atomic state

eg. Mg2+, Mn2+, Cu2+, Pb2+ Flame not dissociate samples into free atoms,ie. PO4

2- interfere Ca2+

analysis (forms CaPO4complex)

Overcome adding cations compete with Ca for P

Ionisation atoms upon dissociation by flame,overcome reducing flame temp.

Matrix interference,eg. atoms in organic solvents enhanced lightabsorption. Overcome pretreat sample (extraction)


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Fluorometry

Measure concentration of solution thatcontain fluorescing molecules

Principle: is based on an energy exchange

process that occurs when valence shellelectrons absorb EMR, become excited andreturn to an energy level lower than theiroriginal level. The lifetime of an excited state

is about 10-9 to 10-6 seconds and the lightemitted fro a single excited state is calledfluorescence


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Monochromator

Primary filter selects the wavelength Secondary filter as in AAS protects the

photodetector from radiant energy

emitted by flourescing molecules insample Spectrofluorometer filters are

replaced with prisms or gratings


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Advantages

Specificity- because selection optimalwavelength for both absorption &fluorescence

Sensitivity- 1000x more sensitive thanspectrophotometry


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Disadvantages

Sensitiveto environmental changes pHchanges

=> affects availability e-

Temperaturechanges=> affects probability loss energy viacollision (rather than fluorescence)

Contaminating chemicals / change solvent=> change structure molecules


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QC Fluorometry

Any fluorescence as result ofenvironmental changes

Quenching

QC: extreme care mandatory=> analytical technique

=> instrument maintenance


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Chemiluminescence

Chemiluminescence differs from flourescence in thatemission of light is created from a chemical orelectrochemical reaction (rxn) and not from EMRstimulation of electrons.

It involves the oxidation of an organic compound suchas luminol, in the presence of a catalyst such as anenzyme, metal ions (though not always)

The excited products formed during the oxidation

rxn produce chemiluminescence on return to thesinglet state that can be measured by a luminometer.

ADVANTAGES


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ADVANTAGESCHEMILUMINESCENCE

Subpicomolar detection limits

Speed rapid

Ease of use, ie. one-step procedure

Simple instrumentation

Increased sensitivity over flourescence


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TURBIDITY & NEPHELOMETRY

Techniques used to measuring the [ ] of a solutionthat contains particles too large for absorptionspectoscopy.

Nephelometry measurement of light scattered by a

particulate solution. Commonly used for antibody-antigen rxns.TURBIDIMETRY measurement of the reduction inlight transmission caused by particle formation.

Applications: microbiology bacterial growth in brothcultures; hematology clot formation in coagulationanalysers.


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QC

Reagents free particles

Cuvettes no scratches Sample handlingcritical because particles

tend aggregate & settle out solution


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Electrochemistry

Measurement of current of voltagegenerated by the activity of specificions

Clinical chemistry: potentiometry;coulometry; voltammetry; andamperometry

All use eithergalvanic electrochemicalcellOR electrolytic cell

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GALVANIC & ELECTROLYTIC


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GALVANIC & ELECTROLYTICCELLS

Electrochemical cell consists of:

Two half-cells and a salt bridge(textbook figure)

Electrodes (cathode & anode) immersed2 beakers salt solution

If only 1 beaker then solution = saltbridge

ION-SELECTIVE


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ION-SELECTIVEELECTRODES (ISE)

Potentiometric method pH electrodes

Sensitive to individuals ions measuredirect electrical potential due toactivity of free ions


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pH Electrodes

Components Indicator Electrode

consists of a silver wirecoated with AgCl in

0.1mmol/L HCl

All above place in into atube containing specialglass membrane tip sensitive to H+ only.


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pH METER

Measures the acidity of a solution.

pH = - log aH+

pH = - log [H+]

AH+ = hydrogen ion activity

[H+] in moles / of solution


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pH METER

2 electrodes measure voltage

1 electrode is in a liquid with fixed acidityreference electrode

Other electrode responds to acidity of thesolution sensing electrode


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pH METER

A voltmeter measures the differencebetween the voltage of the electrodesA meter converts this into pH


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Indicator Electrode

pH electrode into test solution=> movement H+ near tip electrode=> produce potential difference

=> between internal & test solution=> measured as pH by voltmeter


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Reference electrode

Most commonly used - calomel electrode Calomel is a paste of mercurous chloride &

potassium chloride

In electrolyte solution KCl it is in directcontact with metallic mercury All reference electrodes must generate a

stable electrical potential

[ ] of electrolyte must constant &temperature stable voltage


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NERNST EQUATION

Electromotive force generated because H+ atglass tip=> described by Nernst equation (self study)

THUS temperature H+ activity Set temperature-compensation knob

pH COMBINATION


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pH COMBINATIONELECTRODE

Indicator + reference electrodecombine in one small probe

Consists of: internal reference

electrode=> Ag/AgCl OR=> Hg/Hg2Cl2

Sealed into narrow glass cylinder withpH-sensitive glass tip

E E


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pH METER

Combination probe contains both electrodes

H l d


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QC pH electrode

Balance the system with the electrodesin a buffer whose pH is 7.0

Replace buffer with one of different

pH, usually 4.0 or 10DONT touch glass bulb with your fingers.Rinse with distilled water

Keep within ambient temperature rangeAvoid air bubblesKeep clean of deposit

GAS-SENSING


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GAS SENSINGELECTRODES

Similar pH electrodes but separated fromsolution bygas-permeable hydrophobicmembrane

BUT designed detect specific gases insolutions

eg. CO2 (PCO2 electrode)eg. O2 (Clark electrode)eg. NH

3

(NH3

gas electrode)

El h i


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Electrophoresis

Migration charged solutesin electricalfield

In clin lab protein serum, urine,

CSF Lately, Nucleic acids

C


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Components

Electrical power => driving force Support medium

Buffer

Sample

Detecting system

SUPPORT MATERIAL


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SUPPORT MATERIAL

All gels transparent Scanned densitometer Dried permanent record

Cellulose acetate Agarose gel Polyacrylamide gel Starch gel

PRINCIPLE


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PRINCIPLE

Charged particles migrate to oppositecharged electrode

Velocity of migrationcontrolled by=> particle net charge(directly )=> particle size & shape(inversely )=> strength electrical field=> chemical & physical properties supporting

medium=> temperature

P d


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Procedure

Support with gel placed inelectrophoresis chamber

Chamber filled buffer- contact both

ends support/gel Samplesapplied to gel Apply constant voltage / current

specific time - Electrophoresis

D t ti


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Detection

Support/gel in fixative / dried F: prevent diffusion sample

Stainwith appropriate dye aidvisualisation & quantitation

NOTE: dye uptake sample conc.

Excess dye washed away Drygel (permanent record)

QC l t h i


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QC electrophoresis

Operate either constant current / constantvoltage - constant currentpreferred

Why? Current flows through medium heat is

produced Results increased agitation of dissolved

solutes increased current increased heat &buffer evaporation ionic strength of buffer

increased current

QC b ff


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QCbuffer

Affects charge ampholytes (e.g Protein)1 => pH &2 => ionic strength of buffer

Ampholyte net charge either (+) / (-)

if buffer more acidic than pI ampholyte=> ampholyte binds H+

=> ie. (+) charge=> migrate cathode (-)

if buffer more basic than pI ampholyte

=> ampholyte loses H+=> ie. (-) charge=> migrate anode (+)


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DETECTION &


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DE E ION &QUANTITATION

Separated protein fractions stained visualise- UV light (nucleic acids)

Quantitation:densitometer

=> each band = peak=> surface area of peak = % of total


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Ch m t h


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Chromatography

Refers to a group of techniques used toseparate complex mixtures on the basisof different physical interactions

between the individual compound andstationary phase of the system

C mp n nts


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Components

Mobile phase=> gas / liquidF: carry sample

Stationary phase => solid/liquid

F: mobile phase flows

Column: F: hold stationary phase &separated components

MODES OF SEPARATION


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MODES OF SEPARATION

Adsorption chromatography Partition chromatography

Steric exclusion chromatography

Ion-exchange chromatography

Adsorption chromatography


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Adsorption chromatography

Liquid-solidchromatography Competition sample & mobile phasefor

adsorptive sites solid stationary phase High affinity molecules retained longer Stationary phase:(a) acidic polar (eg. silicagel) (b) basic polar (eg. alumina)(c) nonpolar (eg. charcoal)

Partition chromotography


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Partition chromotography

Liquid-liquidchromatography Separation solute basis relative solubility

nonpolar (organic) solvent & polar (aqueous)solvent

Molecules polar & nonpolar groups in aqueoussolution added to immiscible organic solvent

Vigorous shaking -two phases separate

Chloroform method DNA extraction

Steric Exclusion


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Steric Exclusion

Liquid-solidchromatography Separate solute molecules basis of size& shape


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Ion-exchange


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gchromatography

Solute mixtures separated by charge Stationary phase is a resin consistingof large polymers with charged

functional groups Cation exchange resin,anion exchangeresin or mixed bed exchange resin

Resin is insoluble in water

USES OF ION-EXCHANGE


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CHROMATOGRAPHY Removeinterfering substancesfrom

solution

Concentratedilute ion solutions

Separatemixtures charged molecules(eg. amino acids)

Chromotographic procedures


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Chromotographic procedures

1. THIN-LAYER CHROMATOGRAPHY (TLC) Variant column chromatography Thin layer sorbenteg. alumina, silica gel, cellulose, cross-linked

dextran=> uniformly coated glass / plastic plate

Sample applied near bottom edge plate Mobile phase / solventin closed container until atmosphere

saturated solvent vapour Bottom edge plate in solvent NOTE: samples NOT immersed solvent

solvent migrates up thin layer=> capillary action=> sample molecules dissolved=> ie. carries sample molecules

TLC


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TLC

Separation depends sorbent & solvent(1) adsorption(2) partition(3) steric exclusion

(4) ion-exchange Solvent close top => plate removed & dried Sample Rfcompared standards Rf- Rf=>

retention factor Rf = distance sample component

total distance solvent front Method semiquantitativescreening test

HPLC


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HPLC

Improved TLC Components: Pumpforces mobile phase

through column much greater velocity thangravity-flow

Column: stationary phase packed into longstainless steel column

Fine & uniform packing=> high resolution separation=> requires pressure to force mobile phasethrough

Components HPLC


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Components HPLC

Sample injector- small syringe introduce sample intopath

Mobile phase carries sample through column

Detector- monitor eluateas leaves column produce

electronic signal conc. each component Spectrophotometersmost common

Recorder- record detector signal versus time mobilephase ie. from time sample injection

Graph => chromatogram

Chromatograph


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Chromatograph

Identify compounds=> comparedretention timeto standard retentiontimes (BUT only identical conditions)

Determine conc. each compound=> peak area conc. compound

GAS CHROMATOGRAPHY


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GAS CHROMATOGRAPHY

Separate mixtures volatilecompounds / made volatile Similar HPLC except mobile phase = gas Thus samples partitioned between

gaseous mobile phase & liquid stationary phase

Carrier gas=> nitrogen / helium / argon, selectiondepends on type of detector used Samplemust be injected asgas OR Temperatureof the injection port must be above

boiling point of the components to vaporise sampleupon injection

SCINTILLATION COUNTER


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SCINTILLATION COUNTER

Radioimmunoassay used to measuretrace concentrations of hormones ordrugs

Detect radioactive signals Development of non-isotopic

immunoassay diminished use

Osmometry


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Osmometry

Measure conc. solute particles in solution Refers to measurement of osmolality of an aqueous

solution such as serum, plasma or urine 4 physical properties solution change as number

dissolved particles in solvent: collectively they arecalled colligative properties of the solution becausethey can be related to each other and to theosmolality(1) osmotic pressure; (2) vapour pressure; (3)

boiling point; (4) freezing point Thus, osmometry is based on measuring changes inthe colligative properties and the freezing-pointdepression is the most commonly used in clin

FREEZING-POINT


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OSMOMETER Consists of a sample refrigerated chamber containing a stirrer

and a thermistor (temperature sensing device)

Sample is supercooled below its freezing point in a chamberusually containing ethylene glycol

The stirrer is used to agitate the sample in order to initiate

freezing As the ice crystals form, heat is released from the solution,

which at some point reaches an equilibrium with the rate of heatremoved by the colder temp. of the sample chamber

The equilibrium temp is known as the freezing point and is

detected by the thermistor osmolality of the sample and isexpressed as milliosmoles per kilogram of water (mOsm/kg)


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Enzyme Action:


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Induced Fit Model

Enzyme structure flexible, not rigid

Enzyme and active site adjust shape to bind

substrate

Increases range of substrate specificity

Shape changes also improve catalysis duringreaction

Enzyme Action:


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Induced Fit Model

E + S ES complexE + P

SP

P

SS

Learning Check E1


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Learning Check E1

A. The active site is(1) the enzyme(2) a section of the enzyme(3) the substrate

B. In the induced fit model, the shape of

the enzyme when substrate binds(1) Stays the same(2) adapts to the shape of thesubstrate

Factors Affecting Enzyme


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Action: Substrate

Increasing substrate

concentration increases the

rate of reaction (enzyme

concentration is constant)

Maximum activity reachedwhen all of enzyme

combines with substrate

First order kinetics rxn

rate a [substrate]

Zero order kinetics

rxndepends on [enzyme]

Michael is-Menten constant (Km )


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Michael is Menten constant (Km )

1913 Michaelis & Mentenhypothesised role [S]

S binds free E at low [S] (ie. more E than S)

reaction rate steadily as more S added

thus reaction rate [S]=> f i rst-order kinet ics

eventually E saturated with S

thus maximum reaction velocity

as P formed free E immediately combines excess free S=> zero-order k inet ics

thus reaction rate depends [E]

Factors Affecting EnzymeT


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Action: Temperature

Little activity at low temperature Rate increases with temperature

- Movement of molecules

- Rate of intermolecular collusion- Energy for rxn

Most active at optimum temperatures(usually 37C in humans)

Activity lost with denaturation at hightemperatures

Factors Affecting EnzymeA i T


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Action: Temperature low temp. (eg. refrigeration / freezing)

=> enzymes reversible inactive (specimens for enzymeanalysis frozen or refrig.)=> some enzymes NOT frozen (activity lost)=> avoid repeated freeze-thaw (denature)

control temp. lab=> accurate 0.1C

labs choose enzyme analysis=> 25C=> 30C

=> 37C(most common)NOTE: reference ranges vary...

Factors Affecting EnzymeA i


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Action

Optimum temperature

ReactionRate

Low High

Temperature

Factors Affecting Enzyme

A i H


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Action: pH

Maximum activity at optimum pH

R groups of amino acids have proper charge

Tertiary structure of enzyme is correct

Narrow range of activity

Most lose activity in low or high pH

Factors Affecting Enzyme Action


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Factors Affecting Enzyme Action

Reaction

Rate

Optimum pH

3 5 7 9 11

pH

Enzyme Inhibition


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Enzyme Inhibition

Inhibitors

cause a loss of catalytic activity

Change the protein structure of an enzyme May be competitive or noncompetitive

Some effects are irreversible

Competitive Inhibition


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Competitive Inhibition

A competitive inhibitor Has a structure similar to substrate

Occupies active site

Competes with substrate for activesite

Has effect reversed by increasing

substrate concentration


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Learning Check E2


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Learning Check E2

Identify each statement as describing an

inhibitor that is

(1) Competitive (2) Noncompetitive

A. Increasing substrate reverses inhibition

B. Binds to enzyme, not active site

C. Structure is similar to substrate

D. Inhibition is not reversed with substrate

Solution E2


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Solution E2

Identify each statement as describing an

inhibitor that is

(1) Competitive (2) Noncompetitive

A. 1 Increasing substrate reverses inhibition

B. 2 Binds to enzyme, not active site

C. 1 Structure is similar to substrate

D 2 Inhibition is not reversed with substrate

Documents

Unit One Instruments